In the present renewal, our broad objective is to further characterize the MHC class I pathway of antigen processing. We have 3 specific Aims that follow the generation of antigenic peptides from synthesis through antigen processing. The goal of our first aim is to determine the origin (source) of MHC class I presented peptides. We will determine whether the class I pathway is predominantly surveying newly synthesized antigens (neosynthesis) versus the established proteome and critically test the DRiP and immunoribosome hypotheses. As part of our experimental approach we test key predictions of these models by exploiting intein- based catalysis to generate the same antigen either by translation (neosynthesis) or post-translational mechanisms. The goal of our second aim is to definitively determine the role of immunoproteasomes in MHC class I presentation and immune responses. Our previous studies have shown that proteasomes are the key protease needed to make the initial cleavages for the generation of a majority of MHC class I-presented peptides. We and others have shown that there is a special form of proteasomes, called immunoproteasomes, that contain 3 different subunits, each of which encodes an active site. The incorporation of these subunits into proteasomes alters the way in which antigens are cleaved into peptides. Immunoproteasomes are constitutively expressed in dendritic and lymphoid cells and are induced in all other cells by proinflammatory cytokines (e.g. during infection). Our underlying hypothesis is that immunoproteasomes play a particularly important role in generating immunogenic peptides and in so doing play a dominant role in controlling the specificity of CD8 T cell immune responses. It has not been possible to resolve this important issue because up until now, no one has generated animals that lack all three subunits; we have made substantial progress in doing so. Our experimental approach to test our hypothesis is to generate and characterize mutant mice that totally lack immunoproteasomes. The goal of our third Aim is to elucidate the post-proteasomal proteolytic steps in cells that are involved in MHC class I antigen processing. Our underlying hypothesis is that there are several peptidases with distinct but overlapping specificities that shape the repertoire of presented peptides. Our experimental approach to test this hypothesis is to generate and characterize mutant mice that lack individual or combinations of peptidases. We will also test in these knock out systems panels of antigenic substrates in which flanking residues are systematically varied and correlate the results obtained in vivo with those obtained in cell free biochemical assays with recombinant peptidases. Public Health Relevance: This proposal seeks to elucidate how the immune system is able to detect virally infected cells and cancers. This is an important issue because this process is essential to our ability to eliminate or prevent these pathological processes. The information gained by the proposed studies may lead to a better ability to predict and monitor immune responses to viruses and cancers and ultimately aid in the development of vaccines for these diseases.